Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/2292—Thymosin; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• the present invention relates to the field of healing or preventing inflammatory degenerative, immunological and other disorders of the skin and surrounding tissue that occur due to Epidermolysis Bullosa, and all of its subtypes.
• EB Epidermolysis Bullosa
• EB is a rare genetic disorder that afflicts all ethnic and racial groups.
• EB is a group of diseases characterized by blister formation after minor trauma to the skin. This may lead to open sores, ulcerations and scars.
• This family of disorders range in severity from mild to the severely disabling, mutilating and life threatening diseases of the skin. Unlike burns, these afflictions sometimes never go away. The most severe cases require great lifestyle adjustments.
• Afflicted children should never ride a bike, skate, or participate in sports, because the normal play of children causes chronic sores. Such sores may cover as much as 75 percent of the child's body. Blistering and scarring also occur in the mouth and esophagus. Therefore, frequently a diet of only liquids or soft foods is possible. Scarring also causes the fingers and toes to fuse, leaving deformities which severely limit function.
• EB EB Simplex
• Dystrophic EB dominant or recessive
• Junctional EB The severity of symptoms varies between these types.
• EB causes blisters which may be restricted to specific areas, for example hands or feet, or may affect large areas of the body.
• the blisters heal normally without leaving permanent damage to the skin.
• the blisters heal with scarring which can result in permanent change to the skin, for example fingers may fuse and hands contract, reducing movement.
• Some forms of Junctional EB are life threatening in infancy.
• EB results from deleterious changes in the physiological, biochemical and immunological properties of the skin. All forms of EB are genetic in origin and the genes responsible for several different subtypes of the condition are now known. The genetic defects result in the skin layers not adhering properly to each other, causing areas of structural weakness. This fragile skin is particularly vulnerable to damage from mild friction, causing the blisters which are the characteristic feature of the condition. Skin is an important barrier to infection, it is the first line of defense of the immune system. The fragile skin of those afflicted with EB loses this important defense mechanism. Such changes in vasculature decrease capacity to repair damage, increase propensity for skin cancers such as squamous cell carcinoma, and increase risk of infection. In addition, the open sores in the oral and digestive cavity can lead to increased dehydration and malnutrition.
• a method of treatment for promoting healing or prevention of blisters, sores or skin degeneration associated with EB involves administration to a subject or patient in need of such treatment an effective amount of a composition comprising an EB-inhibiting polypeptide comprising amino acid sequence
• the present invention is based on a discovery that actin-sequestering peptides such as thymosin ⁇ 4 (T ⁇ 4) and other actin-sequestering peptides or peptide fragments containing amino acid sequence LKKTET or conservative variants thereof, promote healing or prevention of blisters, sores and skin degeneration associated with
• Epidermolysis Bullosa Included are N- or C-terminal variants such as KLKKTET and LKKTETQ.
• these peptides may have the capacity to promote repair, healing and prevention by having the ability to induce terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines or chemokines, and to act as a chemotactic and/or angiogenic factor for endothelial cells and thus heal and prevent degenerative changes in the skin of patients with afflicted EB, even though EB is the result of an inherited defect.
• terminal deoxynucleotidyl transferase a non-template directed DNA polymerase
• Thymosin ⁇ 4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin ⁇ 4 was originally isolated from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization.
• the invention is a method of treatment for promoting healing and prevention of blisters, sores and skin degradation associated with EB comprising administering to a subject in need of such treatment an effective amount of a composition comprising an EB-inhibiting peptide comprising amino acid sequence LKKTET, or a conservative variant thereof having EB-inhibiting activity, preferably Thymosin ⁇ 4, an isoform of Thymosin ⁇ 4, oxidized Thymosin ⁇ 4, Thymosin ⁇ 4 sulfoxide, or an antagonist of Thymosin ⁇ 4.
• a composition comprising an EB-inhibiting peptide comprising amino acid sequence LKKTET, or a conservative variant thereof having EB-inhibiting activity, preferably Thymosin ⁇ 4, an isoform of Thymosin ⁇ 4, oxidized Thymosin ⁇ 4, Thymosin ⁇ 4 sulfoxide, or an antagonist of Thymosin ⁇ 4.
• LKKTET or conservative variants thereof, having EB-inhibiting activity.
• International Application Serial No. PCT/US99/17282 discloses isoforms of T ⁇ 4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof having EB-inhibiting activity, which may be utilized with the present invention.
• International Application Serial No. PCT/GB99/00833 discloses oxidized Thymosin ⁇ 4 which may be utilized in accordance with the present invention.
• the invention provides a method for healing and preventing blisters and sores of skin in a subject by contacting the skin with an EB-inhibiting effective amount of a composition which contains T ⁇ 4 or a T ⁇ 4 isoform.
• the contacting may be topically or systemically.
• topical administration include, for example, contacting the skin with a lotion, salve, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, or oil comprising T ⁇ 4, alone or in combination with at least one agent that enhances T ⁇ 4 penetration, or delays or slows release of T ⁇ 4 peptides into the area to be treated.
• Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular or subcutaneous injections, or inhalation, transdermal or oral administration of a composition containing T ⁇ 4 or a T ⁇ 4 isoform, etc.
• a subject may be a mammal, preferably human.
• T ⁇ 4 may be administered in any suitable EB-inhibiting amount.
• T ⁇ 4 may be administered in dosages within the range of about 0.1-50 micrograms of T ⁇ 4, more preferably in amounts of about 1-25 micrograms.
• a composition in accordance with the present invention can be administered daily, every other day, etc., with a single administration or multiple administrations per day of administration, such as applications 2, 3, 4 or more times per day of administration.
• T ⁇ 4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of T ⁇ 4.
• Such isoforms include, for example, T ⁇ 4 ala , T ⁇ 9, T ⁇ 10, T ⁇ 11 , T ⁇ 12, T ⁇ 13, T ⁇ 14 and T ⁇ 15. Similar to T ⁇ 4, the T ⁇ 10 and T ⁇ 15 isoforms have been shown to sequester actin.
• T ⁇ 4, T ⁇ 10 and T ⁇ 15, as well as these other isoforms share an amino acid sequence, LKKTET, that appears to be involved in mediating actin sequestration or binding.
• the activity of T ⁇ 4 isoforms may be due, in part, to the ability to regulate the polymerization of actin.
• T ⁇ 4 can modulate actin polymerization in skin (e.g. ⁇ -thymosins appear to depolymerize F-actin by sequestering free G-actin).
• T ⁇ 4's ability to modulate actin polymerization may therefore be due to all, or in part, its ability to bind to or sequester actin via the LKKTET sequence.
• T ⁇ 4 other proteins which bind or sequester actin, or modulate actin polymerization, including T ⁇ 4 isoforms having the amino acid sequence LKKTET, are likely to reduce EB, alone or in a combination with T ⁇ 4, as set forth herein.
• known T ⁇ 4 isoforms such as T ⁇ 4 ala , T ⁇ 9,
• T ⁇ 10, T ⁇ 11 , T ⁇ 12, T ⁇ 13, T ⁇ 14 and T ⁇ 15, as well as T ⁇ 4 isoforms not yet identified, will be useful in the methods of the invention.
• T ⁇ 4 isoforms are useful in the methods of the invention, including the methods practiced in a subject.
• the invention therefore further provides pharmaceutical compositions comprising T ⁇ 4, as well as T ⁇ 4 isoforms T ⁇ 4 ala , T ⁇ 9, T ⁇ 10, T ⁇ 11 , T ⁇ 12, T ⁇ 13, T ⁇ 14 and T ⁇ 15, and a pharmaceutically acceptable carrier.
• proteins having actin sequestering or binding capability or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET, for example, can similarly be employed in the methods of the invention.
• proteins include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin, propo yosin, fincilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, ⁇ -actinin and acumentin, for example.
• the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D binding protein
• the invention includes the use of an EB- inhibiting polypeptide comprising the amino acid sequence LKKTET (which may be within its primary amino acid sequence) and conservative variants thereof.
• LKKTET amino acid sequence LKKTET
• conservative variants thereof denotes the replacement of an amino acid residue by another, biologically similar residue.
• Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
• T ⁇ 4 has been localized to a number of tissue and cell types and thus, agents which stimulate the production of T ⁇ 4 can be added to or comprise a composition to effect T ⁇ 4 production from a tissue and/or a cell.
• agents include members of the family of growth factors, such as insulin-like growth factor (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF- ⁇ ), basic fibroblast growth factor (bFGF), thymosin ⁇ 1 (T ⁇ 1) and vascular endothelial growth factor (VEGF).
• IGF-1 insulin-like growth factor
• PDGF platelet derived growth factor
• EGF epidermal growth factor
• TGF- ⁇ transforming growth factor beta
• bFGF basic fibroblast growth factor
• T ⁇ 1 thymosin ⁇ 1
• VEGF vascular endothelial growth factor
• T ⁇ 4 compositions of the invention may reduce the affects of EB by effectuating growth of the connective tissue through
• subjects are treated with an agent that stimulates production in the subject of an EB-inhibiting peptide as defined above.
• agents that assist or stimulate EB reduction may be added to a composition along with T ⁇ 4 or a T ⁇ 4 isoform.
• agents include angiogenic agents, growth factors, agents that direct differentiation of cells, agents that promote migration of cells and agents that stimulate the provision of extracellular matrix material in the skin.
• T ⁇ 4 or a T ⁇ 4 isoform alone or in combination can be added in combination with any one or more of the following agents: VEGF, KGF, FGF, PDGF, TGF ⁇ , IGF-1 , IGF-2, IL-1 , prothymosin ⁇ and thymosin ⁇ 1 in an effective amount.
• the invention also includes a pharmaceutical composition comprising a therapeutically effective amount of T ⁇ 4 or a T ⁇ 4 isoform in a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier include those listed above with reference to parenteral administration.
• the actual dosage or reagent, formulation or composition that heals or prevents blisters, sores and skin degeneration associated with EB may depend on many factors, including the size and health of a subject.
• persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use.
• Suitable topical formulations include T ⁇ 4 or a T ⁇ 4 isoform at a concentration within the range of about 0.001 - 10% by weight, more preferably within the range of about 0.01 - 0.1 % by weight, most preferably about 0.05% by weight.
• the therapeutic approaches described herein involve various routes of administration or delivery of reagents or compositions comprising the T ⁇ 4 or other compounds of the invention, including any conventional administration techniques (for example, but not limited to, topical administration, local injection, inhalation, or systemic administration), to a subject.
• the methods and compositions using or containing T ⁇ 4 or other compounds of the invention may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers.
• the invention includes use of antibodies which interact with T ⁇ 4 peptide or functional fragments thereof.
• Antibodies which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
• Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art as disclosed in
• antibody as used in this invention is meant to include monoclonal and polyclonal antibodies.
• the invention provides a method of treating a subject by administering an effective amount of an agent which modulates T ⁇ 4 gene expression.
• modulate refers to inhibition or suppression of T ⁇ 4 expression when T ⁇ 4 is over expressed, and induction of expression when T ⁇ 4 is under expressed.
• effective amount means that amount of T ⁇ 4 agent which is effective in modulating T ⁇ 4 gene expression resulting in reducing the symptoms of the T ⁇ 4 associated EB.
• An agent which modulates T ⁇ 4 or T ⁇ 4 isoform gene expression may be a polynucleotide for example.
• the polynucleotide may be an antisense, a triplex agent, or a ribozyme.
• an antisense directed to the structural gene region or to the promoter region of T ⁇ 4 may be utilized.
• the invention provides a method for utilizing compounds that modulate T ⁇ 4 activity.
• Compounds that affect T ⁇ 4 activity include peptides, peptidomimetics, polypeptides, chemical compounds, minerals such as zincs, and biological agents.
• the present invention may promote healing or prevention of blisters, sores and skin degeneration associated with Epidermolysis Bullosa by inducing terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines, or chemokines, and to act as a chemotactic factor for endothelial cells, and thereby promoting healing or preventing degenerative changes in skin brought about by Epidermolysis Bullosa or other degenerative or environmental factors.
• terminal deoxynucleotidyl transferase a non-template directed DNA polymerase

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• 2002
  • 2002-05-16 JP JP2002588889A patent/JP2004525988A/ja active Pending
  • 2002-05-16 CN CNB028100956A patent/CN1241636C/zh not_active Expired - Fee Related
  • 2002-05-16 EP EP02736866A patent/EP1404268A4/en not_active Withdrawn
  • 2002-05-16 CA CA002446072A patent/CA2446072A1/en not_active Abandoned
  • 2002-05-16 WO PCT/US2002/015394 patent/WO2002091969A1/en not_active Ceased
  • 2002-05-16 MX MXPA03010446A patent/MXPA03010446A/es not_active Application Discontinuation
  • 2002-05-16 AU AU2002309842A patent/AU2002309842B2/en not_active Ceased

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